Injury and inflammation trigger the generation of proteases from the circulation, inflammatory cells and epithelial tissues that cleave protease-activated receptors (PARs) to control hemostasis, inflammation, pain and repair. This proposal tests the hypothesis that proteases generated during inflammation cleave PAR2 on sensory nerves. PAR2 activates signaling mechanisms that sensitize ion channels, resulting in release of neuropeptides, which cause neurogenic inflammation and hyperalgesia to inflammatory, thermal, mechanical and osmotic stimuli. These mechanisms will be studied in the intestine, the richest source of proteases in health and diseases states, where neuronal sensitization causes inflammation, pain and functional disturbances by poorly understood mechanisms. They also will be studied in the skin, where proteases induce neurogenic inflammation and pain by well-defined mechanisms due to sensitization of specific ion channels. Studies of transfected cell lines, isolated neurons and genetically-modified mice are proposed. Aim 1 uses PAR2 deficient mice and protease inhibitors to determine the contribution of PAR2 and endogenous proteases to inflammation and pain. The role of protein kinase C epsilon and D in PAR2-induced sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), a major mediator of inflammatory and thermal pain, will be determined using isolated cells and intact animals. Aim 2 defines the signaling pathway by which PAR2 sensitizes TRPV4, and uses TRPV4-deficient mice to determine the role of this channel in inflammation and pain to mechanical and osmotic stimuli. Aim 3 investigates mechanism by which trafficking of PAR2 and TRPV1 to and from the plasma membrane controls neuronal responsiveness. The role of arrestin and ubiquitin in endocytosis and lysosomal trafficking of PAR2, and the role of rabl 1a in mobilization of Golgi PAR2 will be studied in nociceptive neurons. The mechanism by which sensitizing agents, such as PAR2, promote TRPV1 exocytosis, and by which desensitizing agents, such as capsaicin, promote TRPV1 endocytosis and degradation will be examined. An understanding of how proteases regulate neurons will provide new insights into the general mechanisms by which inflammation shapes neuronal plasticity to cause dysfunction and disease, and may lead to new therapies to control neuronal hypersensitivity, inflammation, pain and functional disturbances of the gastrointestinal tract.